Original article
Activation of Akt/mTOR pathway in a patient with atypical teratoid/rhabdoid tumor

Introduction

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant childhood brain tumor, almost inevitably associated with dismal prognosis [17]. AT/RT can occur anywhere in the central nervous system (CNS), including the spinal cord. About 60% of tumors are located in the posterior cranial fossa (especially in the cerebellum). Most rhabdoid tumors were shown to contain chromosome 22 mutation in the region of hSNF5/INI1 gene [2]. The protein encoded by INI1/hSNF5 is a component of the chromatin remodeling SWI/SNF complex, and is a critical tumor suppressor biallelically inactivated in rhabdoid tumors [9]. Identification of INI1 as a tumor suppressor in AT/RT has facilitated diagnosis of rhabdoid tumors.
Although the presence of hSNF5/INI1 mutations in rhabdoid tumors is now well documented, little is known of actual pathophysiology of these neoplasms. However, in a recent study performed on cell lines originating from AT/RT and malignant rhabdoid tumor, Arcaro et al. [1] evaluated the status of several receptor tyrosine kinases. We found that insulin receptor (InR) and insulin-like growth factor-I receptor (IGFIR) are overexpressed in AT/RT cell lines.
This observation turned our attention to the mTOR (mammalian target of rapamycin) pathway. mTOR is a central regulator of cellular state, whose function largely depends on the state of well-being of the cell. If the cell lacks energy, oxygen or nutrients, the kinase inhibits protein synthesis and stops proli-feration. On the other hand, when all these elements are available, cell cycle is triggered with eventual division. Because of its central regulatory role, mTOR kinase is often implicated in tumorigenesis [3,10,18]. As mTOR is directly activated by Akt [4] and Erk [15], we decided to evaluate these two kinases and their pathways in AT/RT.
As material we used tumor mass from the area of left cerebellopontine angle excised from a 3-year old boy with histopathological confirmation of AT/RT.

Material and methods

Tissue samples and sample preparation

AT/RT sample of the tumor excised from the patient as well as control tissues (normal human brain and subependymal giant cell astrocytoma, SEGA) were retrieved from the Department of Patho­logy, Children’s Memorial Hospital, Warsaw, Poland. SEGA sample was retrieved from a TS patient diagnosed clinically according to the criteria of Roach. Control brain tissue consisted of periventricular regions of patients operated on for epilepsy.
For electrophoresis, tissues were homogenized in tissue grinder with RIPA lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS) with 50 mM sodium fluoride and 1 mM sodium orthovanadate, supplemented with 1 × Complete Protease Inhibitor (Roche, Indianapolis, IN) and Phosphatase Inhibitor Cocktail I (Sigma-Aldrich, St. Louis, MO). In order to minimize differences in sample preparation procedure, all the samples were processed at the same time, in the same conditions. Lysates were stored at –80°C.

Western blot

20 µg of protein extracted from tissues or cells were subjected to SDS PAGE in a 10% polyacrylamide gel. Gels were transferred onto PVDF membranes. After blocking with 5% non-fat dry milk in TBST (Tris buffered saline, 0.05% Tween), the blots were incubated with respective primary and secondary (HRP-conjugated) antibodies. Membranes were washed in TBST buffer and proteins were detected by West Pico chemiluminescence substrate (Pierce, Rocford, IL). Even protein loading was verified by Ponceau S staining. Antibodies against: phosphatase and tensin homolog (PTEN), phospho-S6K1 T389, phospho-Erk Y204, phospho-Akt S473, insulin receptor  (InR) and secondary antibodies (HRP-goat anti-rabbit) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phospho-phosphoinositide-dependent kinase-1 (p-PDK1) S241, phospho-glycogen synthase kinase 3 (p-GSK-3) S9, phospho-c-Raf (p-c-Raf) S338, phospho-mitogen-activated protein kinase kinase (p-MEK) S217/S221, phospho-S6 ribosomal protein (p-S6rp) S235/S236 were from Cell Signaling Technologies (Beverly, MA).

Immunohistochemistry

The paraffin sections were routinely stained with haematoxylin-eosin (H&E) and Gomori’s method. Immunohistochemical analyses were performed on paraffin-embedded specimens according to the labelled streptavidin-biotin complex method with DAB as chromogen. S100 protein, epithelial membrane antigen (EMA), glial fibrillary acidic protein (GFAP), synaptophysin and secondary antibodies (HRP-goat anti-rabbit) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclo-nal to SNF5 (ab42503) was purchased from Abcam. AE1/AE3 cytokeratin produced by DAKO was used too.

Results

Histopathologic findings

The tumor consisted of poorly differentiated cells with high mitotic activity. Some cells with abundant pale eosinophilic cytoplasm and eccentrically situated nuclei containing prominent nucleoli were found. The cytoplasm of these cells revealed the presence of globular inclusions. The tumor contained also spindle – cell component and a cell small embryonal element. Large areas of necrosis were encountered.

AT/RT tumor shows unique protein expression pattern

First we evaluated histopathological status of the tumor. We detected extensive staining for S-100 protein normally found in cells of neural crest origin. Then we showed that the tumor also stained well for EMA, whose expression is found in AT/RT by some authors [20], while others do not detect it [12]. Some cells revealed immunoexpression for cytokeratin and GFAP. The focal expression of synaptophysin was also observed. All tumour cells showed loss of expression of SNF5 in nuclei. The intratumour blood cells were positive for SNF5.

Activation of Akt by insulin receptor pathway

Our primary goal was to evaluate whether AT/RT tumor development might be potentiated by the upregulation of mTOR pathway and two kinases leading to mTOR phosphorylation i.e. Akt and Erk. We found that both Akt and its effector, GSK-3, are markedly upregulated. Also PDK1, upstream from Akt, is activated. As Akt pathway is usually triggered by the stimulation of growth factor receptors, we checked and confirmed increased levels of InR. On the other hand, we found that PTEN phosphatase, inhibiting signal transduction to Akt, was not elevated and did not influence activity of this pathway.

Normal levels of phosphorylated Erk

Another kinase sometimes implicated in mTOR-dependent tumor development is Erk. In our case, however, we were unable to confirm activation of any of kinases belonging to Erk cascade. Neither c-Raf, MEK nor Erk itself were active and could lead to mTOR phosphorylation.

Strong signaling to mTOR leads to protein synthesis

As mTOR is a central regulator of protein synthesis, phenomenon responsible for mTOR implication in tumorogenesis, we tested activity of its downstream effectors. Assembly of multisubunit eukaryotic translation initiation factor (eIF4F) complex is inhibited by a family of repressor polypeptides, eIF4E-binding proteins (4E-BPs). 4E-BP1 (also known as PHAS-1) binds eIF4E, inhibiting cap-dependent translation. Incre­ased phosphorylation of 4E-BP1 at S65/T70 disrupts this binding and allows cap-dependent translation. In our AT/RT case we demonstrated upregulation of phosphorylated 4E-BP1. S6K1 kinase phosphorylates S6 ribosomal protein, which is thought to be required for biosynthesis of the translational apparatus. In the tumor both S6K1 and S6rp were hyper-phosphorylated compared to normal human brain.

Discussion

Although it seems that unique disruption of cellular pathways characterising AT/RT may already be established and connected with chromatin remodeling by the SWI/SNF (SWItch/Sucrose NonFermen­table) complex, we are still far from understanding all the events necessary for AT/RT development. It is thought that one of the targets of SWI/SNF complex, including INI1 protein (whose gene is often mutated in AT/RT), may be cyclin D1, responsible for the control of G1/S transition. Normal activity of SWI/SNF complex disrupts cyclin D1 transcription. According to ­others, SWI/SNF complex activates transcription of p16 protein, an inhibitor of cyclin D1. In both cases, when the complex does not work, this leads to uncontrolled acceleration of cell cycle.
Tumorogenic transformation is never a result of a single event. Thus, in the current study we focused on activation of mTOR pathway, which is often found implicated in other brain tumors of the childhood, like medulloblastoma, glioblastoma subependymal giant-cell astrocytoma. Indeed, we found that downstream effectors of mTOR, including 4E-BP1 and S6K1, are upregulated and lead to translation of proteins. As mTOR controls synthesis of essential proteins involved in cell cycle progression, such as cyclin D1 [14], ornithine decarboxylase [16] or survival, like c-Myc [15], its inhibition was found to suppress prolife­ration of cells in all those applications that involved local or systemic control of cell multiplication. mTOR-inhibitor therapies are currently introduced into preclinical models of brain tumors [11].
mTOR is controlled by several upstream mechanisms, responding to stimuli like hypoxia, nutrient availability and energy insufficiency. However, all these factors lead to mTOR inhibition and cannot participate in neoplasmic transformation. On the other hand, growth factors and mitogens signal to mTOR through PI3K/Akt or Ras/Erk pathways, which potentiate mTOR. In our previous studies we demonstrated that active Erk may trigger tumor development in subependymal giant-cell astrocytoma or medulloblastoma [10,19]. Also in other brain tumors upre­gulation of Erk is well-documented [6-8]. In vie of our negative results, this study is the first one to show that Erk pathway probably does not play a role in AT/RT development.
Also PI3K/Akt are often implicated in tumoroge­nesis, especially affecting CNS [13]. Akt, a downstream effector of PI3K, is a typical activator of mTOR-dependent protein translation, to this extent, that numerous authors write about the “PI3K/mTOR” pathway. Thanks to its strong anti-apoptotic and pro-proliferative functions, Akt is found implicated in the pathogenesis of various human malignancies. Here we report consistent upregulation of Akt pathway elements in the case of AT/RT, showing that targeting this kinase may bring favorable outcome in future molecular therapies.

Acknowledgements

This work was supported by the institutional pro­ject 159/06 from The Children’s Memorial Institute and the project R13001106/2009 from Polish Ministry of Science and Higher Education.

References

 1. Arcaro A, Doepfner KT, Boller D, Guerreiro AS, Shalaby T, Jack­son SP, Schoenwaelder SM, Delattre O, Grotzer MA, Fischer B. Novel role for insulin as an autocrine growth factor for malignant brain tumour cells. Biochem J 2007; 406: 57-66.  
2. Biegel JA, Fogelgren B, Zhou JY, James CD, Janss AJ, Allen JC, Zagzag D, Raffel C, Rorke LB. Mutations of the INI1 rhabdoid tumor suppressor gene in medulloblastomas and primitive neuroectodermal tumors of the central nervous system. Clin Cancer Res 2000; 6: 2759-2763.  
3. Choe G, Horvath S, Cloughesy TF, Crosby K, Seligson D, Palotie A, Inge L, Smith BL, Sawyers CL, Mischel PS. Analysis of the phosphatidylinositol 3’-kinase signaling pathway in glioblastoma patients in vivo. Cancer Res 2003; 63: 2742-2746.  
4. Dan HC, Sun M, Yang L, Feldman RI, Sui XM, Ou CC, Nellist M, Yeung RS, Halley DJ, Nicosia SV, Pledger WJ, Cheng JQ. Phosphatidylinositol 3-kinase/Akt pathway regulates tuberous sclerosis tumor suppressor complex by phosphorylation of tuberin. J Biol Chem 2002; 277: 35364-35370.  
5. De Benedetti A, Joshi B, Graff JR, Zimmer SG. CHO cells transformed by the translation factor eIF-4E display increased c-myc expression, but require overexpression of Max for tumorigeni­city. Mol Cell Diff 1994; 2: 347-371.  
6. Dworakowska D, Wlodek E, Leontiou CA, Igreja S, Cakir M, Teng M, Prodromou N, Góth MI, Grozinsky-Glasberg S, Gueorguiev M, Kola B, Korbonits M, Grossman AB. Activation of RAF/MEK/ERK and PI3K/AKT/mTOR pathways in pituitary adenomas and their effects on downstream effectors. Endocr Relat Cancer 2009; 16: 1329-1338.  
7. Forshew T, Tatevossian RG, Lawson AR, Ma J, Neale G, Ogunkolade BW, Jones TA, Aarum J, Dalton J, Bailey S, Chaplin T, Carter RL, Gajjar A, Broniscer A, Young BD, Ellison DW, Sheer D. Activation of the ERK/MAPK pathway: a signature genetic defect in posterior fossa pilocytic astrocytomas. J Pathol 2009; 218: 172-181.  
8. Hagemann C, Gloger J, Anacker J, Said HM, Gerngras S, Kühnel S, Meyer C, Rapp UR, Kämmerer U, Vordermark D, Flentje M, Roosen K, Vince GH. RAF expression in human astrocytic tumors. Int J Mol Med 2009; 23: 17-31.  
9. Isakoff MS, Sansam CG, Tamayo P, Subramanian A, Evans JA, Fillmore CM, Wang X, Biegel JA, Pomeroy SL, Mesirov JP, Roberts CW. Inactivation of the Snf5 tumor suppressor stimulates cell cycle progression and cooperates with p53 loss in oncogenic transformation. Proc Natl Acad Sci USA 2005; 102: 17745-17750.
10. Jozwiak J, Grajkowska W, Kotulska K, Jozwiak S, Zalewski W, Zajaczkowska A, Roszkowski M, Slupianek A, Wlodarski P. Brain tumor formation in tuberous sclerosis depends on Erk activation. Neuromolecular Med 2007; 9: 117-127.
11. Lee N, Woodrum CL, Nobil AM, Rauktys AE, Messina MP, Dabora SL. Rapamycin weekly maintenance dosing and the potential efficacy of combination sorafenib plus rapamycin but not atorvastatin or doxycycline in tuberous sclerosis preclinical models. BMC Pharmacol 2009; 9: 8.
12. Makuria AT, Rushing EJ, McGrail KM, Hartmann DP, Azumi N, Ozdemirli M. Atypical teratoid rhabdoid tumor (AT/RT) in adults: review of four cases. J Neurooncol 2008; 88: 321-330.
13. McBride SM, Perez DA, Polley MY, Vandenberg SR, Smith JS, Zheng S, Lamborn KR, Wiencke JK, Chang SM, Prados MD, Ber­ger MS, Stokoe D, Haas-Kogan DA. Activation of PI3K/mTOR pathway occurs in most adult low-grade gliomas and predicts patient survival. J Neurooncol 2010; 97: 33-40.
14. Rosenwald IB, Kaspar R, Rousseau D, Gehrke L, Leboulch P, Chen JJ, Schmidt EV, SonenbergN, London IM. Eukaryotic translation initiation factor 4E regulates expression of cyclin D1 at transcriptional and post-transcriptional levels. J Biol Chem 1995; 270: 21176-21180.
15. Roux PP, Ballif BA, Anjum R, Gygi SP, Blenis J. Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase. Proc Natl Acad Sci USA 2004; 101: 13489-13494.
16. Shantz LM, Pegg AE. Overproduction of ornithine decarboxylase caused by relief of translational repression is associated with neoplastic transformation. Cancer Res 1994; 54: 2313-2316.
17. Squire SE, Chan MD, Marcus KJ. Atypical teratoid/rhabdoid tumor: the controversy behind radiation therapy. J Neurooncol 2007; 81: 97-111.
18. Wlodarski PK, Boszczyk A, Grajkowska W, Roszkowski M, Jozwiak J. Implication of active Erk in the classic type of human medulloblastoma. Folia Neuropathol 2008; 46: 117-122.
19. Włodarski P, Grajkowska W, Łojek M, Rainko K, Jóźwiak J. Activation of Akt and Erk pathways in medulloblastoma. Folia Neuropathol 2006; 44: 214-220.
20. Wu X, Dagar V, Algar E, Muscat A, Bandopadhayay P, Ashley D, Wo Chow C. Rhabdoid tumour: a malignancy of early childhood with variable primary site, histology and clinical behaviour. Pathology 2008; 40: 664-670.